Molecular genetic analysis of colon cancers has established that the Wnt signaling pathway is involved in early tumor development in the intestine. The transcription factors that commute Wnt signals into changes in target gene expression are members of the Lymphoid Enhancer Factor/T Cell Factor (LEF/TCF) family. There are full-length activating and truncated dominant negative forms of LEF/TCFs as well as alternatively spliced isoforms. The activities of LEF/TCFs are modified by signaling pathways that cross-talk through co-regulation of target genes, and by pathways that modify the actions of specific LEF/TCF isoforms. Two pathways have been discovered to be important in this regard. First, the Notch pathway collaborates with Wnt signaling to maintain a stem cell niche and actively cycling progenitor cells in the intestine. This collaboration may be important in colon cancer to maintain proliferating cancer-initiating cells (stem or progenitor cells). Second, a kinase cascade that involves calcium-calmodulin Kinase II (CAMKII) modifies LEF/TCF localization and activity. Our data shows that TCF-1 localization is dramatically altered in human colon cancer. The following hypotheses are proposed. First, the hypothesis that Notch and CAMKII are active signals in colon cancer will be tested in primary human colon cancer and newly derived colon cancer initiating cell cultures (CCICs) (Aim1). Second, the hypothesis that Notch signals modify Wnt target expression will be tested by blocking Notch signaling in colon cancer and CCIC cultures. Additional emphasis will be placed on an analysis of LEF1 and its activities with Notch signaling components (Aim2). Third, the hypothesis that CAMKII causes nuclear export of a dominant negative TCF-1 isoform to enhance Wnt signaling will be tested in colon cancer cells and in biochemical assays. The biological consequences of dnTCF-1 nuclear exclusion will be assessed by doxycycline-induced shRNA knockdown (Aim3). The overall goal of these projects are to determine whether Notch cross-talk and CAMKII signaling enhance Wnt signals to promote tumorigenesis, or moderate the pathway to maintain cancer-initiating stem cells. PUBLIC HEALTH RELEVANCE: This project proposes to define how a signal transduction pathway that is important for normal intestine function (Wnt), is altered to promote cancer. In the intestine, Wnt signals control stem cells, cell proliferation and mature cell function. Other pathways cooperate with Wnts to ensure that the intestine maintains proper these growth patterns. Wnt signals are abnormally strong in cancer, and these same collaborating pathways may be helping push cells into a tumorigenic state. This project examines how two such collaborating pathways (Notch and Calmodulin Kinase II) modify Wnt signals in colon cancer and in newly established colon cancer stem cell lines.